Mechanisms of the Asc1 amino acid transporter
[eng] The Asc1 amino acid transporter is involved in several basic processes of the central nervous system, such as N-methyl-D-aspartate receptor mediated excitation and glycinergic inhibition of signal transmission, having thus potential as a therapeutic target to treat neurological disorders such...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2024 |
| País: | España |
| Institución: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/216040 |
| Acceso en línea: | https://hdl.handle.net/2445/216040 http://hdl.handle.net/10803/692397 |
| Access Level: | acceso abierto |
| Palabra clave: | Ciències de la salut Aminoàcids Microscòpia electrònica Transport biològic Medical sciences Amino acids Electron microscopy Biological transport |
| Sumario: | [eng] The Asc1 amino acid transporter is involved in several basic processes of the central nervous system, such as N-methyl-D-aspartate receptor mediated excitation and glycinergic inhibition of signal transmission, having thus potential as a therapeutic target to treat neurological disorders such as schizophrenia, amyotrophic lateral sclerosis and hyperekplexia. In the present project, we aimed to determine the high-resolution atomic structure of Asc1 in complex with CD98hc using Cryo-EM and to dissect the molecular mechanisms that underlie its transport function, therefore laying the groundwork for future research to develop more efficient drugs against the aforementioned neurological disorders. The structure of the Asc1/CD98hc complex has been determined at a resolution of 4 Ǻ, with local resolution reaching 3.4-3.8 Ǻ for most of the map. The transporter has been captured in a hitherto unknown semi- occluded conformation, in which the transmembrane helix (TM) 1a is in an intermediate position between a transporter fully open to the cytosol and one completely occluded. Interestingly, in this apo structure a connection can be observed between the bundle (TM1, 2, 6, 7) and scaffold (TM3, 4, 5, 8, 9, 10) domains through a polar contact between Ser 246 (TM6) and Tyr 333 (TM8). Recalling previous studies with prokaryotic homologues, which proved the importance of this kind of interactions between domains for substrate translocation, the functional relevance of the Ser 246 – Tyr 333 was studied. Although the Cryo-EM structure was obtained from a protein preparation incubated with 10 mM D-serine the resulting structure was in a substrate- free apo state. To surmount this limitation, Protein Energy Landscape Exploration (PELE) was carried out with the newly determined Asc1 structure, allowing us to identify a series of residues key for determining substrate selectivity in Asc1: Ser 56, Ser 246 and Tyr 333. The impact of mutating these residues on the uptake of radiolabelled substrate was measured, revealing that all three residues are essential for the transport of the canonical Asc1 substrates, i. e., small neutral amino acids, but not larger substrates. Moreover, the impact of mutating these residues on both modes of transport presented by Asc1, exchange and facilitated diffusion, was assessed. Efflux experiments in HeLa cells with the mutants S246G and Y333F revealed that these residues are essential for exchange, but also result in a slower inward return of the empty transporter, thus supporting also a role of Ser 246 and Tyr 333 in sustaining facilitated diffusion. Molecular dynamics of L-alanine binding in our structure largely confirmed the observations from the PELE analysis although, surprisingly, in one of the trajectories TM1a opened the cytosolic vestibule and the substrate was released to the cytosol. In this replica, initially Tyr 333 competed with Ser 56 for binding with the substrate carboxyl. The rotation of Tyr 333 towards TM6 dragged the substrate away from the unwound region of TM1, and upon disconnection of the substrate from TM1, TM1a started to open the cytosolic gate. The binding of the side chain of Ser 246 to L-alanine, which we had also observed in one of the PELE poses, could be stabilising an intermediate stage in which Tyr 333 has rotated towards TM6 but the substrate remains bound to Ser 56. We propose a model in which the interaction of the substrate with the side chains of Ser 246 and Tyr 333 allows its proper positioning between the unwound regions of TM1 and TM6 to trigger translocation. This model is supported by kinetic analysis of L-alanine and D-serine uptake of the mutants S246G and Y333F, which decrease the maximal velocity of the transporter. |
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